The applicant's goal is to understand how cells can generate asymmetry and coordinate differentiation with cell cycle progression using the simple model bacterium, C. crescentus. In C. crescentus, the predivisional cell is polarized with a stalk at one pole and a single flagellum at the other pole. Every cell cycle includes an asymmetric division that gives rise to two morphologically and physiologically different daughter cells. The system affords access to genetics, biochemistry, genomics and new cytology tools to look at protein dynamics in live cells. A complex phosphorelay of two-component signal transduction proteins is at the heart of differentiation and cell cycle control in this organism. This project has three objectives: The first objective is to sort out the interactions and functions of several of the two-component proteins that participate in cell cycle control and differentiation. To do this, the investigators will use gene expression profiling, phosphorylation assays as well as cell imaging technology. The second objective is based on the recent observations that several components of this regulatory network exhibit a dynamic behavior of spatial localization, alternating between dispersed distribution and discrete accumulation at the cell pole in a cell cycle-dependent manner. To understand how their cell cycle spatial localization relates to regulation and function, the applicants will determine cis-acting sequences and factors that control polar localization of these signaling proteins. The third objective is to identify new cell cycle regulators using a genetic approach. The ultimate goal is to dissect in time and space the signal transduction mechanisms of the two-component regulatory network that controls the C. crescentus differentiation and cell cycle. Components of this essential cell cycle regulatory network are conserved among medically important microorganisms. Insights gained into the cellular organization of prokaryotes and the mechanisms used by them to control temporal and spatial processes will not only close a gap in our understanding of fundamentals of bacterial physiology and regulation, but will also provide a basis for rational design of new antibacterial agents. [unreadable] [unreadable]